Antimicrobial composition

ABSTRACT

The present invention relates to novel antimicrobial composition comprising a peptide which can be obtained from the milk protein casein or chemically synthesised or produced by recombinant DNA technology and a divalent cation.

FIELD OF THE INVENTION

The present invention relates to novel antimicrobial compositioncomprising a peptide which can be obtained from the milk protein caseinor chemically synthesised or produced by recombinant DNA technology anda divalent cation. These compositions can be used in foods asantimicrobial preservatives, in oral care products (eg. toothpaste,mouthwash, dental floss) for the control of dental plaque andsuppression of pathogens associated with dental caries and periodontaldiseases.

BACKGROUND OF THE INVENTION

Kappacin, the nonglycosylated, phosphorylated forms of bovinecaseinomacropeptide (CMP), has been shown to have antibacterial activityin vitro against both Gram-negative and Gram-positive oral bacteria(Malkoski et al., 2001). CMP is a 64 amino acid polypeptide releasedfrom bovine κ-casein by chymosin hydrolysis of the peptide bond betweenPhe¹⁰⁵ and Met¹⁰⁶. It comprises the 106-169 C-terminal fragment ofκ-casein and contains all the post-translational modification sitesfound in κ-casein. CMP is both variably phosphorylated and glycosylated(Pisano et al., 1984; Saito and Itoh, 1992; Talbo et al., 2001). CMP iscompletely phosphorylated at Ser¹⁴⁹ and partially phosphorylated (10%)at Ser¹²⁷ as determined by MALDI-PSD mass spectrometry (Talbo et al.,2001). Additionally there are at least six genetic variants of κ-casein,with variants A and B being by far the most common (Creamer and Harris,1997). Variants A and B differ at residues 136 and 148where thehydrophilic residues Thr¹³⁶ and Asp¹⁴⁸ of variant A are substituted bythe hydrophobic residues Ile¹³⁶ and Ala¹⁴⁸ in variant B. Theantibacterial active region of Kappacin was demonstrated to be residues138-158 as determined using the synthetic peptideSer(P)¹⁴⁹κ-casein-A(138-158). Phosphorylation of Ser¹⁴⁹ was shown to beessential for antibacterial activity using the synthetic peptideκ-casein-A(138-158) (Malkoski et al., 2001). The MIC of CMP variant Aagainst Streptococcus mutans 0.68 mg/ml (100 μM) whilst variant B wasless active with a MIC of 1.04 mg/ml (154 μM) (Malkoski et al., 2001).

The mechanism by which Kappacin inhibits bacterial growth is stillunclear. Kappacin was found to be most effective against S. mutans atslightly acidic growth pH. The non-glycosylated, κ-casein-B(130-158) hasbeen proposed to form an amphipathic α-helix, especially in the presenceof trifluoroethanol (TFE; Plowman, 1997). This characteristic could helpto explain its antibacterial activity if it works as a surface-activeagent, creating pores in the cell membrane. This mode of action has beenproposed for the majority of the cationic antimicrobial peptidesisolated to date. However Kappacin is an anionic peptide that does notexhibit sequence similarity with the better known cationic antibacterial peptides and apart from a possible propensity to form anamphipathic helical structure does not posses any of the othercharacteristics of these peptides. Kappacin does share somecharacteristics with the recently discovered anionic antibacterialpeptides, especially enkelytin. This peptide, like Kappacin, contains anumber of glutamyl residues and phosphorylation is essential forantibacterial activity (Goumon, 1996; Goumon, 1998; Strub, 1996). Thestructure of the phosphorylated form of enkelytin has not beendetermined, although phosphorylation has been proposed to change theconformation of the peptide through electrostatic repulsion or bydivalent metal ion binding (Goumon, 1998; Kieffer, 1998). It remainsunclear how the negatively charged antibacterial peptides, includingKappacin, interact with the bacterial cell surface.

SUMMARY OF THE INVENTION

The present inventors investigated the effect of pH and divalent metalcations on the antibacterial activity and structure of the peptide. Thepresent inventors were able to show a synergistic effect between thepeptides and divalent cations.

Accordingly, in a first aspect the present invention consists in anantimicrobial composition, the composition comprising a divalent cationand a peptide, the peptide being non-glycosylated, less than about 100amino acids, preferably less than about 70 amino acids, and comprisingan amino acid sequence selected from the group consisting of:—

(SEQ ID NO: 1) Ala Val Glu Ser Thr Val Ala Thr Leu Glu Ala Ser(P) ProGlu Val Ile Glu Ser Pro Pro Glu, (SEQ ID NO: 2) Ala Val Glu Ser Thr ValAla Thr Leu Glu Asp Ser(P) Pro Glu Val Ile Glu Ser Pro Pro Glu,and conservative substitutions therein.

In a preferred embodiment of the present invention the peptide comprisesan amino acid sequence selected from the group consisting of:—

(SEQ ID NO: 1) Ala Val Glu Ser Thr Val Ala Thr Leu Glu Ala Ser(P) ProGlu Val Ile Glu Ser Pro Pro Glu, and (SEQ ID NO: 2) Ala Val Glu Ser ThrVal Ala Thr Leu Glu Asp Ser(P) Pro Glu Val Ile Glu Ser Pro Pro Glu.

In a further preferred embodiment of the present invention the peptidecomprises an amino acid sequence selected from the group consisting of:—

(SEQ ID NO: 3) Met Ala Ile Pro Pro Lys Lys Asn Gln Asp Lys Thr Glu IlePro Thr Ile Asn Thr Ile Ala Ser Gly Glu Pro Thr Ser Thr Pro Thr Ile GluAla Val Glu Ser Thr Val Ala Thr Leu Glu Ala Ser(P) Pro Glu Val Ile GluSer Pro Pro Glu Ile Asn Thr Val Gln Val Thr Ser Thr Ala Val; (SEQ ID NO:4) Met Ala Ile Pro Pro Lys Lys Asn Gln Asp Lys Thr Glu Ile Pro Thr IleAsn Thr Ile Ala Ser(P) Gly Glu Pro Thr Ser Thr Pro Thr Ile Glu Ala ValGlu Ser Thr Val Ala Thr Leu Glu Ala Ser(P) Pro Glu Val Ile Glu Ser ProPro Glu Ile Asn Thr Val Gln Val Thr Ser Thr Ala Val; (SEQ ID NO: 5) MetAla Ile Pro Pro Lys Lys Asn Gln Asp Lys Thr Glu Ile Pro Thr Ile Asn ThrIle Ala Ser Gly Glu Pro Thr Ser Thr Pro Thr Thr Glu Ala Val Glu Ser ThrVal Ala Thr Leu Glu Asp Ser(P) Pro Glu Val Ile Glu Ser Pro Pro Glu IleAsn Thr Val Gln Val Thr Ser Thr Ala Val; (SEQ ID NO: 6) Met Ala Ile ProPro Lys Lys Asn Gln Asp Lys Thr Glu Ile Pro Thr Ile Asn Thr Ile AlaSer(P) Gly Glu Pro Thr Ser Thr Pro Thr Thr Glu Ala Val Glu Ser Thr ValAla Thr Leu Glu Asp Ser(P) Pro Glu Val Ile Glu Ser Pro Pro Glu Ile AsnThr Val Gln Val Thr Ser Thr Ala Val; (SEQ ID NO: 7) Thr Glu Ile Pro ThrIle Asn Thr Ile Ala Ser Gly Glu Pro Thr Ser Thr Pro Thr Ile Glu Ala ValGlu Ser Thr Val Ala Thr Leu Glu Ala Ser(P) Pro Glu Val Ile Glu Ser ProPro Glu Ile Asn Thr Val Gln Val Thr Ser Thr Ala Val; (SEQ ID NO: 8) ThrGlu Ile Pro Thr Ile Asn Thr Ile Ala Ser(P) Gly Glu Pro Thr Ser Thr ProThr Ile Glu Ala Val Glu Ser Thr Val Ala Thr Leu Glu Ala Ser(P) Pro GluVal Ile Glu Ser Pro Pro Glu Ile Asn Thr Val Gln Val Thr Ser Thr Ala Val;(SEQ ID NO: 9) Thr Glu Ile Pro Thr Ile Asn Thr Ile Ala Ser Gly Glu ProThr Ser Thr Pro Thr Thr Glu Ala Val Glu Ser Thr Val Ala Thr Leu Glu AspSer(P) Pro Glu Val Ile Glu Ser Pro Pro Glu Ile Asn Thr Val Gln Val ThrSer Thr Ala Val; (SEQ ID NO: 10) Thr Glu Ile Pro Thr Ile Asn Thr Ile AlaSer(P) Gly Glu Pro Thr Ser Thr Pro Thr Thr Glu Ala Val Glu Ser Thr ValAla Thr Leu Glu Asp Ser(P) Pro Glu Val Ile Glu Ser Pro Pro Glu Ile AsnThr Val Gln Val Thr Ser Thr Ala Val;and conservative substitutions therein.

It is further preferred that the peptide comprises an amino acidsequence selected from the group consisting of:—

(SEQ ID NO: 3) Met Ala Ile Pro Pro Lys Lys Asn Gln Asp Lys Thr Glu IlePro Thr Ile Asn Thr Ile Ala Ser Gly Glu Pro Thr Ser Thr Pro Thr Ile GluAla Val Glu Ser Thr Val Ala Thr Leu Glu Ala Ser(P) Pro Glu Val Ile GluSer Pro Pro Glu Ile Asn Thr Val Gln Val Thr Ser Thr Ala Val; (SEQ ID NO:4) Met Ala Ile Pro Pro Lys Lys Asn Gln Asp Lys Thr Glu Ile Pro Thr IleAsn Thr Ile Ala Ser(P) Gly Glu Pro Thr Ser Thr Pro Thr Ile Glu Ala ValGlu Ser Thr Val Ala Thr Leu Glu Ala Ser(P) Pro Glu Val Ile Glu Ser ProPro Glu Ile Asn Thr Val Gln Val Thr Ser Thr Ala Val; (SEQ ID NO: 5) MetAla Ile Pro Pro Lys Lys Asn Gln Asp Lys Thr Glu Ile Pro Thr Ile Asn ThrIle Ala Ser Gly Glu Pro Thr Ser Thr Pro Thr Thr Glu Ala Val Glu Ser ThrVal Ala Thr Leu Glu Asp Ser(P) Pro Glu Val Ile Glu Ser Pro Pro Glu IleAsn Thr Val Gln Val Thr Ser Thr Ala Val; (SEQ ID NO: 6) Met Ala Ile ProPro Lys Lys Asn Gln Asp Lys Thr Glu Ile Pro Thr Ile Asn Thr Ile AlaSer(P) Gly Glu Pro Thr Ser Thr Pro Thr Thr Glu Ala Val Glu Ser Thr ValAla Thr Leu Glu Asp Ser(P) Pro Glu Val Ile Glu Ser Pro Pro Glu Ile AsnThr Val Gln Val Thr Ser Thr Ala Val; (SEQ ID NO: 7) Thr Glu Ile Pro ThrIle Asn Thr Ile Ala Ser Gly Glu Pro Thr Ser Thr Pro Thr Ile Glu Ala ValGlu Ser Thr Val Ala Thr Leu Glu Ala Ser(P) Pro Glu Val Ile Glu Ser ProPro Glu Ile Asn Thr Val Gln Val Thr Ser Thr Ala Val; (SEQ ID NO: 8) ThrGlu Ile Pro Thr Ile Asn Thr Ile Ala Ser(P) Gly Glu Pro Thr Ser Thr ProThr Ile Glu Ala Val Glu Ser Thr Val Ala Thr Leu Glu Ala Ser(P) Pro GluVal Ile Glu Ser Pro Pro Glu Ile Asn Thr Val Gln Val Thr Ser Thr Ala Val;(SEQ ID NO: 9) Thr Glu Ile Pro Thr Ile Asn Thr Ile Ala Ser Gly Glu ProThr Ser Thr Pro Thr Thr Glu Ala Val Glu Ser Thr Val Ala Thr Leu Glu AspSer(P) Pro Glu Val Ile Glu Ser Pro Pro Glu Ile Asn Thr Val Gln Val ThrSer Thr Ala Val; and (SEQ ID NO: 10) Thr Glu Ile Pro Thr Ile Asn Thr IleAla Ser(P) Gly Glu Pro Thr Ser Thr Pro Thr Thr Glu Ala Val Glu Ser ThrVal Ala Thr Leu Glu Asp Ser(P) Pro Glu Val Ile Glu Ser Pro Pro Glu IleAsn Thr Val Gln Val Thr Ser Thr Ala Val.

In yet a further preferred embodiment of the present invention thepeptide is selected from the group consisting of:—

(SEQ ID NO: 3) Met Ala Ile Pro Pro Lys Lys Asn Gln Asp Lys Thr Glu IlePro Thr Ile Asn Thr Ile Ala Ser Gly Glu Pro Thr Ser Thr Pro Thr Ile GluAla Val Glu Ser Thr Val Ala Thr Leu Glu Ala Ser(P) Pro Glu Val Ile GluSer Pro Pro Glu Ile Asn Thr Val Gln Val Thr Ser Thr Ala Val; (SEQ ID NO:4) Met Ala Ile Pro Pro Lys Lys Asn Gln Asp Lys Thr Glu Ile Pro Thr IleAsn Thr Ile Ala Ser(P) Gly Glu Pro Thr Ser Thr Pro Thr Ile Glu Ala ValGlu Ser Thr Val Ala Thr Leu Glu Ala Ser(P) Pro Glu Val Ile Glu Ser ProPro Glu Ile Asn Thr Val Gln Val Thr Ser Thr Ala Val; (SEQ ID NO: 5) MetAla Ile Pro Pro Lys Lys Asn Gln Asp Lys Thr Glu Ile Pro Thr Ile Asn ThrIle Ala Ser Gly Glu Pro Thr Ser Thr Pro Thr Thr Glu Ala Val Glu Ser ThrVal Ala Thr Leu Glu Asp Ser(P) Pro Glu Val Ile Glu Ser Pro Pro Glu IleAsn Thr Val Gln Val Thr Ser Thr Ala Val; (SEQ ID NO: 6) Met Ala Ile ProPro Lys Lys Asn Gln Asp Lys Thr Glu Ile Pro Thr Ile Asn Thr Ile AlaSer(P) Gly Glu Pro Thr Ser Thr Pro Thr Thr Glu Ala Val Glu Ser Thr ValAla Thr Leu Glu Asp Ser(P) Pro Glu Val Ile Glu Ser Pro Pro Glu Ile AsnThr Val Gln Val Thr Ser Thr Ala Val; (SEQ ID NO: 7) Thr Glu Ile Pro ThrIle Asn Thr Ile Ala Ser Gly Glu Pro Thr Ser Thr Pro Thr Ile Glu Ala ValGlu Ser Thr Val Ala Thr Leu Glu Ala Ser(P) Pro Glu Val Ile Glu Ser ProPro Glu Ile Asn Thr Val Gln Val Thr Ser Thr Ala Val; (SEQ ID NO:8); ThrGlu Ile Pro Thr Ile Asn Thr Ile Ala Ser(P) Gly Glu Pro Thr Ser Thr ProThr Ile Glu Ala Val Glu Ser Thr Val Ala Thr Leu Glu Ala Ser(P) Pro GluVal Ile Glu Ser Pro Pro Glu Ile Asn Thr Val Gln Val Thr Ser Thr Ala Val;(SEQ ID NO: 9) Thr Glu Ile Pro Thr Ile Asn Thr Ile Ala Ser Gly Glu ProThr Ser Thr Pro Thr Thr Glu Ala Val Glu Ser Thr Val Ala Thr Leu Glu AspSer(P) Pro Glu Val Ile Glu Ser Pro Pro Glu Ile Asn Thr Val Gln Val ThrSer Thr Ala Val; (SEQ ID NO: 10) Thr Glu Ile Pro Thr Ile Asn Thr Ile AlaSer(P) Gly Glu Pro Thr Ser Thr Pro Thr Thr Glu Ala Val Glu Ser Thr ValAla Thr Leu Glu Asp Ser(P) Pro Glu Val Ile Glu Ser Pro Pro Glu Ile AsnThr Val Gln Val Thr Ser Thr Ala Val; (SEQ ID NO: 1) Ala Val Glu Ser ThrVal Ala Thr Leu Glu Ala Ser(P) Pro Glu Val Ile Glu Ser Pro Pro; and (SEQID NO: 2) Ala Val Glu Ser Thr Val Ala Thr Leu Glu Asp Ser(P) Pro Glu ValIle Glu Ser Pro Pro.

The divalent cation is preferably selected from the group consisting ofZn²⁺, Ca²⁺, Cu²⁺, Ni²⁺, Co²⁺, Fe²⁺, Sn²⁺, and Mn²⁺. In addition, thedivalent cation may be in association with fluoride such as SnF⁺ andCuF⁺. It is currently preferred, however, that the divalent cation isCa²⁺ or Zn²⁺.

It is further preferred that the molar ratio of the divalent cation tothe peptide is in the range of 0.5:1.0 to 15.0:1.0, preferably in therange of 0.5:1.0 to 4.0:1.0. It is further preferred that the molarratio of the divalent cation to the peptide is in the range of 1.0:1.0to 4.0:1.0, preferably 1.0:1.0 to 2.0:1.0.

In a still further preferred embodiment the composition furthercomprises a pharmaceutically-acceptable carrier. Such compositions maybe dental, intra-oral compositions, therapeutic anti-infectivecompositions for topical and systemic application. Dental compositionsor therapeutic compositions may be in the form of a gel, liquid, solid,powder, cream or lozenge. Therapeutic compositions may also be in theform of tablets or capsules.

In a further aspect, there is provided a method of treating orpreventing dental caries or periodontal disease in a subject, the methodcomprising the step of administering the composition of the presentinvention to the teeth or gums of a subject in need of such treatments.Topical administration of the composition is preferred.

As it is the physical nature of the peptides rather than the specificsequence of the peptide which results in their antimicrobial activity socalled conservative substitutions may be made in the peptide sequencewith no substantial loss of activity. It is intended that suchconservative substitutions which do not result in a substantial loss ofactivity are encompassed in the present invention.

Whilst the concept of conservative substitution is well understood bythe person skilled in the art, for the sake of clarity conservativesubstitutions are those set out below.

Gly, Ala, Val, Ile, Leu, Met; Asp, Glu, Ser; Asn, Gln; Ser, Thr; Lys,Arg, His; Phe, Tyr, Trp, His; and Pro, Nα-alkalamino acids.

The compositions of the present invention have a number of applications,for example, they can be used in foods as antimicrobial preservatives,in oral care products (toothpastes and mouthrinses) for the control ofdental plaque and suppression of pathogens associated with dental cariesand periodontal diseases. The antimicrobial compositions of the presentinvention may also be used in pharmaceutical preparations (eg, topicaland systemic anti-infective medicines).

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element or integer or group of elements or integers but notthe exclusion of any other element or integer or group of elements orintegers.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an antimicrobial composition comprisinga divalent cation and at least one peptide. These peptides wereinitially derived from casein, κ-casein (106-169) [Table 1].

The peptides Ser(P)¹⁴⁹ κ-casein (117-169) and Ser(P)¹²⁷, Ser(P)¹⁴⁹κ-casein (117-169) can be purified from a tryptic digest of bovinecasein using standard chromatographic procedures of anion exchange andreversed-phase chromatography (HPLC). Ser(P)¹⁴⁹ κ-casein (106-169) andSer(P)¹²⁷, Ser(P)¹⁴⁹ κ-casein (106-169) can also be prepared from cheesewhey and rennet whey by removal of the whey proteins by ultrafiltration,or acid precipitation followed by reversed-phase HPLC purification ofthe phosphopeptides. The peptides can be prepared from casein of otherspecies, eg. goat, sheep etc.

TABLE 1 Casein Antimicrobial Peptides Peptide Sequence^(a) Ser(P)¹⁴⁹κ-casein B (106-169) Met Ala Ile Pro Pro Lys Lys Asn Gln Asp Lys Thr GluIle Pro Thr Ile Asn Thr Ile Ala Ser Gly Glu Pro Thr Ser Thr Pro Thr IleGlu Ala Val Glu Ser Thr Val Ala Thr Leu Glu Ala Ser(P) Pro Glu Val IleGlu Ser Pro Pro Glu Ile Asn Thr Val Gln Val Thr Ser Thr Ala Val (SEQ IDNO: 3) Ser(P)¹²⁷, Ser(P)¹⁴⁹ κ-casein B Met Ala Ile Pro Pro Lys Lys AsnGln Asp Lys Thr (106-169) Glu Ile Pro Thr Ile Asn Thr Ile Ala Ser(P) GlyGlu Pro Thr Ser Thr Pro Thr Ile Glu Ala Val Glu Ser Thr Val Ala Thr LeuGlu Ala Ser(P) Pro Glu Val Ile Glu Ser Pro Pro Glu Ile Asn Thr Val GlnVal Thr Ser Thr Ala Val (SEQ ID NO: 4) Ser(P)¹⁴⁹, κ-casein A (106-169)Met Ala Ile Pro Pro Lys Lys Asn Gln Asp Lys Thr Glu Ile Pro Thr Ile AsnThr Ile Ala Ser Gly Glu Pro Thr Ser Thr Pro Thr Thr Glu Ala Val Glu SerThr Val Ala Thr Leu Glu Asp Ser(P) Pro Glu Val Ile Glu Ser Pro Pro GluIle Asn Thr Val Gln Val Thr Ser Thr Ala Val (SEQ ID NO: 5) Ser(P)¹²⁷,Ser(P)¹⁴⁹ κ-casein A Met Ala Ile Pro Pro Lys Lys Asn Gln Asp Lys Thr(106-169) Glu Ile Pro Thr Ile Asn Thr Ile Ala Ser(P) Gly Glu Pro Thr SerThr Pro Thr Thr Glu Ala Val Glu Ser Thr Val Ala Thr Leu Glu Asp Ser(P)Pro Glu Val Ile Glu Ser Pro Pro Glu Ile Asn Thr Val Gln Val Thr Ser ThrAla Val (SEQ ID NO: 6) Ser(P)¹⁴⁹ κ-casein B (117-169) Thr Glu Ile ProThr Ile Asn Thr Ile Ala Ser Gly Glu Pro Thr Ser Thr Pro Thr Ile Glu AlaVal Glu Ser Thr Val Ala Thr Leu Glu Ala Ser(P) Pro Glu Val Ile Glu SerPro Pro Glu Ile Asn Thr Val Gln Val Thr Ser Thr Ala Val (SEQ ID NO: 7)Ser(P)¹²⁷, Ser(P)¹⁴⁹ κ-casein B Thr Glu Ile Pro Thr Ile Asn Thr Ile AlaSer(P) Gly (117-169) Glu Pro Thr Ser Thr Pro Thr Ile Glu Ala Val Glu SerThr Val Ala Thr Leu Glu Ala Ser(P) Pro Glu Val Ile Glu Ser Pro Pro GluIle Asn Thr Val Gln Val Thr Ser Thr Ala Val (SEQ ID NO: 8) Ser(P)¹⁴⁹κ-casein A (117-169) Thr Glu Ile Pro Thr Ile Asn Thr Ile Ala Ser Gly GluPro Thr Ser Thr Pro Thr Thr Glu Ala Val Glu Ser Thr Val Ala Thr Leu GluAsp Ser(P) Pro Glu Val Ile Glu Ser Pro Pro Glu Ile Asn Thr Val Gln ValThr Ser Thr Ala Val (SEQ ID NO: 9) Ser(P)¹²⁷, Ser(P)¹⁴⁹ κ-casein A ThrGlu Ile Pro Thr Ile Asn Thr Ile Ala Ser(P) Gly (117-169) Glu Pro Thr SerThr Pro Thr Thr Glu Ala Val Glu Ser Thr Val Ala Thr Leu Glu Asp Ser(P)Pro Glu Val Ile Glu Ser Pro Pro Glu Ile Asn Thr Val Gln Val Thr Ser ThrAla Val (SEQ ID NO: 10) Ser(P)¹⁴⁹ κ-casein B (138-158) Ala Val Glu SerThr Val Ala Thr Leu Glu Ala Ser(P) Pro Glu Val Ile Glu Ser Pro Pro (SEQID NO: 1) Ser(P)¹⁴⁹ κ-casein A (138-158) Ala Val Glu Ser Thr Val Ala ThrLeu Glu Asp Ser(P) Pro Glu Val Ile Glu Ser Pro Pro (SEQ ID NO: 2)

The peptide κ-casein (106-169) is present in cheese whey or rennet wheyin several different forms. The peptide has two major genetic variants(A and B) and is post-translationally modified by glycosylation andphosphorylation. The glycosylated forms, known as theKappa-caseino-glycopeptide or glycomacropeptide have been described byNeeser [U.S. Pat. Nos. 4,992,420 and 4,994,441] as anti-plaque andanti-caries agents by virtue of the oligosaccharide chains linked tothreonine residues of the peptide. Neeser claims that theoligosaccharide chains of the glycopeptide, by specifically binding toplaque-forming oral bacteria, block the adherence of these bacteria ontosalivary-coated tooth enamel. The glycosylated forms of κ-casein(106-169) can be separated from the non-glycosylated forms bychromatography (eg. anion exchange and reversed-phase HPLC) or byselective precipitation or ultrafiltration. Only the non-glycosylatedforms of κ-casein (117-169) or κ-casein (106-169) showed antimicrobialactivity. As glycosylation destroys antimicrobial activity it isdesirable to separate the glyco- and aglyco-forms of κ-casein (117-169)or κ-casein (106-169) which can be achieved using chromatography,selective precipitation or ultrafiltration. Phosphorylation of Ser¹⁴⁹and to a lesser extent Ser¹²⁷ are important for antimicrobial activityand the phosphorylated forms of the two major genetic variants (A and B)appear to possess equal activity [Table 1]. The Neeser patents do notdisclose the antimicrobial activity of κ-casein(106-169) nor the use ofthe non-glycosylated forms of the peptide for the suppression ofbacterial pathogens.

In a particularly preferred embodiment of the invention, theantimicrobial composition is incorporated into dentifrices such astoothpaste, mouth washes or formulations for the mouth to aid in theprevention and/or treatment of dental caries and periodontal diseases.The peptide may comprise 0.01-50% by weight of the dentifricecomposition, preferably 0.1-10%. For oral compositions it is preferredthat the amount of the composition of the present invention administeredis 0.01-50% by weight, preferably 0.1-10% by weight of the composition.The oral composition of this invention which contains theabove-mentioned peptides may be prepared and used in various formsapplicable to the mouth such as dentifrice including toothpastes,toothpowders and liquid dentifrices, mouthwashes, troches, chewing gums,dental pastes, gingival massage creams, gargle tablets, lozenges, dairyproducts and other foodstuffs. The oral composition according to thisinvention may further include additional well known ingredientsdepending on the type and form of a particular oral composition.

In certain highly preferred forms of the invention the oral compositionmay be substantially liquid in character, such as a mouthwash or rinse.In such a preparation the vehicle is typically a water-alcohol mixturedesirably including a humectant as described below.

Generally, the weight ratio of water to alcohol is in the range of fromabout 1:1 to about 20:1. The total amount of water-alcohol mixture inthis type of preparation is typically in the range of from about 70 toabout 99.9% by weight of the preparation. The alcohol is typicallyethanol or isopropanol. Ethanol is preferred.

The pH of such liquid and other preparations of the invention isgenerally in the range of from about 4.5 to about 9 and typically fromabout 5.5 to 8. The pH is preferably in the range of from about 6 toabout 8.0, preferably 7.4. The pH can be controlled with acid (e.g.citric acid or benzoic acid) or base (e.g. sodium hydroxide) or buffered(as with sodium citrate, benzoate, carbonate, or bicarbonate, disodiumhydrogen phosphate, sodium dihydrogen phosphate, etc).

Other desirable forms of this invention, the oral composition may besubstantially solid or pasty in character, such as toothpowder, a dentaltablet or a dentifrice, that is a toothpaste (dental cream) or geldentifrice. The vehicle of such solid or pasty oral preparationsgenerally contains dentally acceptable polishing material. Examples ofpolishing materials are water-insoluble sodium metaphosphate, potassiummetaphosphate, tricalcium phosphate, dihydrated calcium phosphate,anhydrous dicalcium phosphate, calcium pyrophosphate, magnesiumorthophosphate, trimagnesium phosphate, calcium carbonate, hydratedalumina, calcined alumina, aluminium silicate, zirconium silicate,silica, bentonite, and mixtures thereof. Other suitable polishingmaterial include the particulate thermosetting resins such as melamine-,phenolic, and urea-formaldehydes, and cross-linked polyepoxides andpolyesters. Preferred polishing materials include crystalline silicahaving particle sized of up to about 5 microns, a mean particle size ofup to about 1.1 microns, and a surface area of up to about 50,000cm²/gm., silica gel or colloidal silica, and complex amorphous alkalimetal aluminosilicate.

When visually clear gels are employed, a polishing agent of colloidalsilica, such as those sold under the trademark SYLOID as Syloid 72 andSyloid 74 or under the trademark SANTOCEL as Santocel 100, alkali metalalumino-silicate complexes are particularly useful since they haverefractive indices close to the refractive indices of gellingagent-liquid (including water and/or humectant) systems commonly used indentifrices.

Many of the so-called “water insoluble” polishing materials are anionicin character and also include small amounts of soluble material. Thus,insoluble sodium metaphosphate may be formed in any suitable manner asillustrated by Thorpe's Dictionary of Applied Chemistry, Volume 9, 4thEdition, pp. 510-511. The forms of insoluble sodium metaphosphate knownas Madrell's salt and Kurrol's salt are further examples of suitablematerials. These metaphosphate salts exhibit only a minute solubility inwater, and therefore are commonly referred to as insolublemetaphosphates (IMP). There is present therein a minor amount of solublephosphate material as impurities, usually a few percent such as up to 4%by weight. The amount of soluble phosphate material, which is believedto include a soluble sodium trimetaphosphate in the case of insolublemetaphosphate, may be reduced or eliminated by washing with water ifdesired. The insoluble alkali metal metaphosphate is typically employedin powder form of a particle size such that no more than 1% of thematerial is larger than 37 microns.

The polishing material is generally present in the solid or pastycompositions in weight concentrations of about 10% to about 99%.Preferably, it is present in amounts from about 10% to about 75% intoothpaste, and from about 70% to about 99% in toothpowder. Intoothpastes, when the polishing material is silicious in nature, it isgenerally present in amount of about 10-30% by weight. Other polishingmaterials are typically present in amount of about 30-75% by weight.

In a toothpaste, the liquid vehicle may comprise water and humectanttypically in an amount ranging from about 10% to about 80% by weight ofthe preparation. Glycerine, propylene glycol, sorbitol and polypropyleneglycol exemplify suitable humectants/carriers. Also advantageous areliquid mixtures of water, glycerine and sorbitol. In clear gels wherethe refractive index is an important consideration, about 2.5-30% w/w ofwater, 0 to about 70% w/w of glycerine and about 20-80% w/w of sorbitolare preferably employed.

Toothpaste, creams and gels typically contain a natural or syntheticthickener or gelling agent in proportions of about 0.1 to about 10,preferably about 0.5 to about 5% w/w. A suitable thickener is synthetichectorite, a synthetic colloidal magnesium alkali metal silicate complexclay available for example as Laponite (e.g. CP, SP 2002, D) marketed byLaporte Industries Limited. Laponite D is, approximately by weight58.00% SiO₂, 25.40% MgO, 3.05% Na₂O, 0.98% Li₂O, and some water andtrace metals. Its true specific gravity is 2.53 and it has an apparentbulk density of 1.0 g/ml at 8% moisture.

Other suitable thickeners include Irish moss, iota carrageenan, gumtragacanth, starch, polyvinylpyrrolidone, hydroxyethylpropylcellulose,hydroxybutyl methyl cellulose, hydroxypropyl methyl cellulose,hydroxyethyl cellulose (e.g. available as Natrosol), sodiumcarboxymethyl cellulose, and colloidal silica such as finely groundSyloid (e.g. 244). Solubilizing agents may also be included such ashumectant polyols such propylene glycol, dipropylene glycol and hexyleneglycol, cellosolves such as methyl cellosolve and ethyl cellosolve,vegetable oils and waxes containing at least about 12 carbons in astraight chain such as olive oil, castor oil and petrolatum and esterssuch as amyl acetate, ethyl acetate and benzyl benzoate.

It will be understood that, as is conventional, the oral preparationsare to be sold or otherwise distributed in suitable labelled packages.Thus, a jar of mouthrinse will have a label describing it, in substance,as a mouthrinse or mouthwash and having directions for its use; and atoothpaste, cream or gel will usually be in a collapsible tube,typically aluminium, lined lead or plastic, or other squeeze, pump orpressurized dispenser for metering out the contents, having a labeldescribing it, in substance, as a toothpaste, gel or dental cream.

Organic surface-active agents are used in the compositions of thepresent invention to achieve increased prophylactic action, assist inachieving thorough and complete dispersion of the active agentthroughout the oral cavity, and render the instant compositions morecosmetically acceptable. The organic surface-active material ispreferably anionic, nonionic or ampholytic in nature which does notdenature the antimicrobial peptide of the invention, and it is preferredto employ as the surface-active agent a detersive material which impartsto the composition detersive and foaming properties while not denaturingthe peptide. Suitable examples of anionic surfactants are water-solublesalts of higher fatty acid monoglyceride monosulfates, such as thesodium salt of the monosulfated monoglyceride of hydrogenated coconutoil fatty acids, higher alkyl sulfates such as sodium lauryl sulfate,alkyl aryl sulfonates such as sodium dodecyl benzene sulfonate, higheralkylsulfo-acetates, higher fatty acid esters of 1,2-dihydroxy propanesulfonate, and the substantially saturated higher aliphatic acyl amidesof lower aliphatic amino carboxylic acid compounds, such as those having12 to 16 carbons in the fatty acid, alkyl or acyl radicals, and thelike. Examples of the last mentioned amides are N-lauroyl sarcosine, andthe sodium, potassium, and ethanolamine salts of N-lauroyl, N-myristoyl,or N-palmitoyl sarcosine which should be substantially free from soap orsimilar higher fatty acid material. The use of these sarconite compoundsin the oral compositions of the present invention is particularlyadvantageous since these materials exhibit a prolonged marked effect inthe inhibition of acid formation in the oral cavity due to carbohydratesbreakdown in addition to exerting some reduction in the solubility oftooth enamel in acid solutions. Examples of water-soluble nonionicsurfactants suitable for use with peptides are condensation products ofethylene oxide with various reactive hydrogen-containing compoundsreactive therewith having long hydrophobic chains (e.g. aliphatic chainsof about 12 to 20 carbon atoms), which condensation products(“ethoxamers”) contain hydrophilic polyoxyethylene moieties, such ascondensation products of poly (ethylene oxide) with fatty acids, fattyalcohols, fatty amides, polyhydric alcohols (e.g. sorbitan monostearate)and polypropyleneoxide (e.g. Pluronic materials).

Surface active agent is typically present in amount of about 0.1-5% byweight. It is noteworthy, that the surface active agent may assist inthe dissolving of the peptide of the invention and thereby diminish theamount of solubilizing humectant needed.

Various other materials may be incorporated in the oral preparations ofthis invention such as whitening agents, preservatives, silicones,chlorophyll compounds and/or ammoniated material such as urea,diammonium phosphate, and mixtures thereof. These adjuvants, wherepresent, are incorporated in the preparations in amounts which do notsubstantially adversely affect the properties and characteristicsdesired.

Any suitable flavouring or sweetening material may also be employed.Examples of suitable flavouring constituents are flavouring oils, e.g.oil of spearmint, peppermint, wintergreen, sassafras, clove, sage,eucalyptus, marjoram, cinnamon, lemon, and orange, and methylsalicylate. Suitable sweetening agents include sucrose, lactose,maltose, sorbitol, xylitol, sodium cyclamate, perillartine, AMP(aspartyl phenyl alanine, methyl ester), saccharine, and the like.Suitably, flavour and sweetening agents may each or together comprisefrom about 0.1% to 5% more of the preparation.

In the preferred practice of this invention an oral compositionaccording to this invention such as mouthwash or dentifrice containingthe composition of the present invention is preferably applied regularlyto the gums and teeth, such as every day or every second or third day orpreferably from 1 to 3 times daily, at a pH of about 4.5 to about 9,generally about 5.5 to about 8, preferably about 6 to 8, for at least 2weeks up to 8 weeks or more up to a lifetime.

The compositions of this invention can be incorporated in lozenges, orin chewing gum or other products, e.g. by stirring into a warm gum baseor coating the outer surface of a gum base, illustrative of which may bementioned jelutong, rubber latex, vinylite resins, etc., desirably withconventional plasticisers or softeners, sugar or other sweeteners orsuch as glucose, sorbitol and the like.

In another embodiment, the composition of the invention is formulated infoods to act as a preservative preferably comprising 0.01-10% w/w, morepreferably 0.1-5% w/w, most preferably 1-5% and particularly 2% w/w.

The present invention provides compositions including pharmaceuticalcompositions comprising the divalent cation and the peptide as describedtogether with a pharmaceutically-acceptable carrier. Such compositionsmay be selected from the group consisting of dental, intra-oralcompositions, therapeutic anti-infective compositions for topical andsystemic application. Dental compositions or therapeutic compositionsrnay be in the form of a gel, liquid, solid, powder, cream or lozenge.Therapeutic compositions may also be in the form of tablets or capsules.

The present invention also provides a method of treating or preventingdental caries or periodontal disease comprising the step ofadministering the composition of the invention to the teeth or gums of asubject in need of such treatments. Topical administration of thecomposition is preferred.

It will be clearly understood that, although this specification refersspecifically to applications in humans, the invention is also useful forveterinary purposes. Thus in all aspects the invention is useful fordomestic animals such as cattle, sheep, horses and poultry; forcompanion animals such as cats and dogs; and for zoo animals.

In order that the nature of the present invention may be more clearlyunderstood preferred forms thereof will now be described with referenceto the following non-limiting examples.

FIGURE LEGENDS

FIG. 1: Chromatogram of purified Ser(P)¹⁴⁹κ-casein-B(138-158). A sampleof the purified peptide fraction was applied to a RP-HPLC analyticalcolumn. Purified peptide was eluted from the column using a lineargradient of 0-100% buffer B (30 min). The flow rate was 1 mL/min. BufferA was 0.1% acetic acid in water, pH 5.5 (TEA) and buffer B was 60%acetonitrile containing 0.1% acetic acid in water, pH 5.5 (TEA).

FIG. 2: Mass spectrometric analysis of RP-HPLC fraction B using theMALDI-TOF MS. The major peak observed with a MW of 2233.9 Dacorresponded to the synthesised peptide, Ser(P)¹⁴⁹ κ-casein-B(138-158).Spectrum was obtained in linear, negative mode with an acceleratingvoltage of 20 kV, grid voltage of 93% and pulse delay time of 100 ns.

FIG. 3. Effect of κ-casein-A(106-169) [▴]; κ-casein-B(106-169) [⋄];ZnCl₂ [X]; Zn:κ-casein-B(106-169) in a 1:1 ratio [▪] andZn:κ-casein-A(106-169) in a 1:1 ratio [♦] on Streptococcus mutans growthin THYE at pH 7.2.

FIG. 4. Effect of calcium ion concentration on the growth inhibitoryactivity of 250 μM κ-casein-A(106-169) tested against S. mutans at pH7.2. CaCl₂ control (♦); Ca:κ-casein-A(106-169) (▴). κ-casein-A(106-169)was incubated with CaCl₂ at ratios between 1:0 and 1:4 for 1 h prior toaddition to the assay.

FIG. 5. Scatchard analysis of zinc binding to κ-casein-A(106-169). ZnCl₂was incubated with purified κ-casein-A(106-169) in water at pH 7.3 for 1h at 37° C. Samples were then centrifuged through 3,000 molecular weightcut-off filtration membranes. The amount of zinc ions was determined byatomic absorption spectrometry.

FIG. 6 Effect of Kappacin (10 mg/ml), ZnCl₂ (20 mM) and Kappacin:ZnCl₂when used as mouthrinses as the only form of oral hygiene on the meanplaque index scores for posterior teeth. a=significantly different fromwater control; b=significantly different from all other treatments; asdetermined using the Wilcoxon rank test.

FIG. 7. Effect of mouthrinses on distribution of plaque index scores ofposterior teeth.

In order that the nature of the present invention may more readilyunderstood preferred forms thereof will now be described with referenceto the following Examples.

MATERIALS AND METHODS Kappacin Preparation

Caseinate-HCl (Bonlac Foods, Melbourne Australia) was dissolved by slowaddition with constant stirring to deionised water at 50° C., pH 8.0 togive a final concentration of 21.5 g/L. Once the caseinate haddissolved, the temperature was lowered to 37° C. and the pH adjusted to6.3 by the slow addition of 1 M HCl to avoid precipitation of casein. Tobegin the hydrolysis Rennet (90% Chymosin EC 3.4.23.4, 145 IMCU/ml,Single Strength, Chr. Hanson) was added to a final concentration of 1.2IMCU/g casein and the solution stirred at 37° C. for 1 h. The pH of thesolution was maintained at 6.3±0.2 by the addition of 1 M HCl and 1 MNaOH. Hydrolysis was stopped by the addition of trichloroacetic acid toa final concentration of 4% and the precipitated proteins were pelletedby centrifugation (5,000 g, 15 min, 4° C.). The supernatant containingthe caseinomacropeptide (CMP) was concentrated by diafiltration using a3000 Da cutoff membrane (S10Y3, Amicon). This material was thenlyophilized. This preparation was further fractionated into glycosylatedforms, non-glycosylated κ-casein-A(106-169) and nonglycosylatedκ-casein-B(106-169) by reversed phase HPLC using a C₁₈ column andelution with 90% acetonitrile/0.1% v/v TFA, as described previously(Malkoski et al., 2001). The eluant was monitored using a primarywavelength of 215 nm. The identity of each fraction was confirmed bymass spectrometric analysis using a Voyager linear matrix assisted laserdesorption/ionisation time of flight mass spectrometer (MALDI MS;PerSeptive Biosystems, MA, USA) and N-terminal sequence analysis asdescribed previously (Malkoski et al., 2001).

Solid Phase Peptide Synthesis and Purification

Peptides corresponding to Ser(P)¹⁴⁹κ-casein-A(138-158) andSer(P)¹⁴⁹κ-casein-B(138-158) were synthesized using standard solid phasepeptide synthesis protocols as described previously (Malkoski et al.,2001). Peptides were purified by reversed phase HPLC using a C₁₈ columnand identified by mass spectrometric analysis as described previously(Malkoski et al., 2001).

Antibacterial Planktonic Assay

The oral opportunistic pathogen Streptococcus mutans Ingbritt was usedin this study as an indicator strain. The antibacterial assay wasconducted in 96 well plates and bacterial growth continuously monitoredover 40 h, as previously described (Malkoski et al., 2001). Briefly,bacteria were cultured in Todd Hewitt Broth (36.4 g/l) containing Yeastextract (5.0 g/l) and 100 mM potassium phosphate with a pH of 6.3 or7.2. The bacterial inocula were prepared by diluting exponentiallygrowing cells in growth medium to give 2.7×10⁴ viable cells/ml. In thebacterial growth assays test wells contained κ-casein-A(106-169) orκ-casein-B(106-169) at concentrations between 20 and 120 μM. thesepeptides were also tested in combination with zinc or calcium ions togive ratios of Kappacin:divalent metal ion of 1:1 to 1:4. SyntheticSer(P)¹⁴⁹κ-casein-A(138-158) and Ser(P)¹⁴⁹κ-casein-B(138-158) peptideswere also tested in this assay. The plates were incubated at 37° C. andgrowth determined by measuring optical density (OD) at 620 nm using aniEMS microplate reader (Labsystems, OY Research Technologies Division).

Biofilm Growth of S. mutans.

A constant depth film fermenter (CDFF: Wimmpenny, Cardiff University,UK) was used for biofilm formation. The CDFF consists of a stainlesssteel disc rotating at a constant speed of 3 rpm containing 15polytetrafluoroethylene (PTFE) pans each of which contain 5 plugs of 4.5mm in diameter. The plugs were set to 0.4 mm below the surface of thesteel disc. Inbuilt scrapers were used to maintain a constant biofilmdepth of 0.4 mm. The scrapers are attached so that the stainless steeldisc rotates under them; the scrapers are spring loaded so that they arepressed down against the pans. An anaerobic atmosphere was maintained inthe CDFF by continuous gassing with 5% CO₂ in N₂. The CDFF was housed ina modified CO₂ incubator, which was used to maintain a constant culturetemperature of 37° C. A S. mutans Ingbritt batch culture in Todd Hewitt(35.4 g/l), Yeast Extract (8 g/L) broth (THYE) in exponential growthphase was used to inoculate the CDFF at a flow rate of 30 ml/h for 5 h.The growth media, THYE containing 0.1% (w/v) sucrose, was then pumpedinto the CDFF at a constant flow rate of 40 ml/h.

At specified times prior to and after treatment with solutions, plugswere removed from the CDFF, washed to remove planktonic bacteria andviable counts were performed to determine bacterial numbers. To assessthe effect on cell viability of various solutions growth media additionto the CDFF was suspended for 10 min and replaced by the solution at aflow rate of 30 ml/h. After 10 min growth media addition was resumed.Solutions tested in the CDFF were: 2 mM Tris-HCl pH6.0; 10 mg/mlKappacin preparation (see above) in 2 mM Tris-HCl pH 6.0; 10 mg/mlKappacin preparation (see above) and 20 mM ZnCl₂ in 2 mM Tris-HCl pH6.0; 20 mM ZnCl₂ in 2 mM Tris-HCl pH 6.0; 2 mM ZnCl₂ in 2 mM Tris-HCl pH6.0 and 0.05% chlorhexidine digluconate in deionized water.

Divalent Metal Cation Binding Assay

CaCl₂ or ZnCl₂ at specified concentrations between 135 and 540 μM wasincubated with purified κ-casein-A(106-169) at a concentration of 135 μMin water at pH 7.3 for 1 h at 37° C. with stirring. Samples were thencentrifuged (1,000 g, 10 min) through 3,000 Da cut-off filtrationmembranes (YM3 Cellulose, Millipore Bedford Mass., USA) to separateunbound divalent cations from the peptide with bound cations. The amountof calcium or zinc ions in the filtrate and initial sample was thendetermined by atomic absorption spectrometry (Model 373 AAS,Perkin-Elmer) set on absorption mode at 422.7 nm or 213.9 nm,respectively. The total amount of zinc or calcium in the sample and free(unbound) zinc or calcium was calculated by reference to a standardcurve. Binding to κ-casein-A(106-169) was determined by Scatchardanalysis.

Structural Determination

One dimensional ¹H NMR spectra of synthetic Ser(P)¹⁴⁹κ-casein-A(138-158)were acquired on a Varian Unity Inova spectrometer (Palo Alto, Calif.,USA) operating at 600 MHz. A series of spectra were recorded at aconstant pH of 6.5 with trifluoroethanol (TFE) concentrations of 0, 5,15 and 30% (v/v) and an initial peptide concentration of 3 mM. The pHwas adjusted by dropwise addition of 1 M HCl. A spectrum was recordedwith a final peptide concentration of 2.3 mM in 30% TFE and 3 mM CaCl₂.All spectra were recorded at a probe temperature of 5° C. Solventsuppression was achieved through the use of the WET-1D sequence(Smallcombe et al., 1995).

Clinical Trial

Ten subjects were recruited for the double-blind, cross-over study.Subjects were recruited from undergraduate students enrolled at theSchool of Dental Science, The University of Melbourne. The groupconsisted of 6 females and 4 males with a mean age of 21 years. Subjectswere examined prior to the commencement of the trial and all were gaugedto be of good health, having dentitions without unrestored cariouslesions or evidence of moderate to severe gingivitis. The subjects hadnot used any dentifrices containing antimicrobial agents prior tocommencement of the trial. Approval was obtained from the Human ResearchEthics Committee of the University of Melbourne.

At the commencement of the trial, the subjects were instructed to ceaseall other forms of daily oral hygiene practices and to solely rely onthe use of the allocated mouthrinse solution. Four solutions were testedas mouthrinses in this investigation: Solution A: Deionized water. B: 1%(w/v) Kappacin preparation in deionized water. C: 20 mM ZnCl indeionized water. D: 1% (w/v) Kappacin preparation and 20 mM ZnCl indeionized water. The pH of all solutions was adjusted to 6.9±0.1 usingKOH. Subjects were instructed to rinse three times daily: morning,mid-day and evening with 15 ml of solution for a duration of one minute.Each trial session was for four days with a clinical evaluation at theend of the trial. The Silness and Loe Plaque Index (PI) was used toevaluate plaque (Silness and Loe, 1964). The gingival area of each toothsurface (distal, buccal, mesial and lingual) was given a score from 0-3.All teeth excluding third molars were scored at the conclusion of thetrial. Following each trial session the subjects resumed their normaloral hygiene habits for a minimum of seven days prior to the next trial.Data were analysed using the nonparametric Wilcoxon rank test.

Results.

Antibacterial Activity of the Genetic Variants of κ-Casein(106-169).

To determine whether the difference in the relative antibacterialactivities of the two major genetic variants of Kappacin[κ-casein(106-169)] activity was due to the amino acid sequencedifference in the previously identified active region ofκ-casein-A(106-169), residues 138-158, Ser(P)¹⁴⁹κ-casein-B(138-158) wassynthesized and its activity tested. The purity of the syntheticSer(P)¹⁴⁹κ-casein-B(138-158) was determined by reversed-phase HPLC and asingle peak was observed (FIG. 1). Analysis of this peak by massspectrometry gave a single peak with an observed mass (m/z) of 2233.9 Dawhich corresponded to the calculated mass for the synthetic peptide,Ser(P)¹⁴⁹κ-casein-B(138-158) of 2235.4 (FIG. 2). The calculated MIC forthe synthetic peptide Ser(P)¹⁴⁹κ-casein-B(138-158) tested against S.mutans at a growth pH of 6.28 in the microplate growth assay was 44 μM.

Interaction of Kappacin with Divalent Metal Cations.

There was no inhibition of S. mutans growth by either of the syntheticactive region peptides [Ser(P)¹⁴⁹κ-casein-A(138-158) andSer(P)¹⁴⁹κ-casein-B(138-158)] or the genetic variants of the purifiedfull length peptides when tested at a growth pH of 7.20 up toconcentrations of 300 μM. When the two genetic variants ofκ-casein(106-169) were tested for bacterial growth inhibitory activityat pH 7.20 in the presence of an equimolar concentration of theantibacterial divalent cation Zn²⁺ a synergistic effect was observed(FIG. 3). Zinc ions alone had a MIC of 200 μM, which masked thesynergistic effect of Kappacin and zinc when tested at ratios above 1:1.Interestingly when the zinc ions were replaced with calcium ions anantibacterial effect was detectable with κ-casein-A(106-169) although noeffect on S. mutans growth could be detected with κ-casein-B(106-169)and calcium in a 1:1 ratio up to 300 μM (Table 2).

The optimal ratio of calcium to κ-casein-A(106-169) for bioactivity wasdetermined by testing various ratios against S. mutans in the microplategrowth assay. A ratio of 2:1 was shown to be more effect than 1:1,whilst increasing the calcium:κ-casein-A(106-169) ratio to 4:1 did notincrease activity (FIG. 4).

Scatchard analysis of binding assays using κ-casein-A(106-169) with thedivalent cation Zn²⁺ demonstrated that there were two binding sites forzinc in this peptide (FIG. 5). Similar results were obtained for calciumbinding.

TABLE 2 Minimal Inhibitory Concentrations of the two genetic variants ofnon-glycosylated, phosphorylated, κ-casein(106-169) and the syntheticpeptide Ser(P)¹⁴⁹κ-casein-A(138-158) tested singly and in combinationwith a 1:1 ratio of zinc or calcium against S. mutans at pH 7.20. MIC(μM) κ-casein-A(106-169) NI* κ-casein-B(106-169) NI κ-casein-A(106-169)and Calcium 248 κ-casein-B(106-169) and Calcium NI κ-casein-A(106-169)and Zinc 161 κ-casein-B(106-169) and Zinc 200Ser(P)¹⁴⁹κ-casein-A(138-158) and Zinc 149 Ser(P)¹⁴⁹κ-casein-A(138-158)NI ZnCl₂ 200 CaCl₂ NI *No inhibition at concentrations up to 1 mMEffect of Kappacin and Zinc on the Viability of Streptococcus mutansCultured as a Biofilm.

After inoculation into the CDFF biofilm fermenter S. mutans rapidlyformed a stable biofilm that contained 5-6×10⁸ viable cells per plug.Addition of 5 ml of 2 mM Tris-HCl pH 6.0 had little effect on the viablecount of S. mutans in the biofilm. In contrast addition of 5 ml of a 1%(w/v) Kappacin preparation in 2 mM Tris-HCl pH 6.0 resulted in a rapiddecrease in the S. mutans viable cell count such that 2 h after additionthere had been a 99.5% reduction in the viable count. Recovery of the S.mutans biofilm was slow after Kappacin addition and 3 days after theaddition bacterial counts were still less than 13% of the pretreatmentlevel. Addition of 5 ml of 2 mM ZnCl₂ in 2 mM Tris-HCl pH 6.0 to astable biofilm of S. mutans in the CDFF reduced the viable count by˜60%. The number of viable cells rapidly recovered from this treatment.The addition of 20 mM ZnCl₂ to the biofilm in an identical mannerresulted in a decrease in viable cell counts of 92% Again a rapidrecovery of viable cell counts was observed. The addition ofKappacin:zinc (1% w/v Kappacin, 20 mM Zn) to a S. mutans biofilm causeda rapid decrease in viable cell numbers, with a 96.0% decrease in 2 h.However, three days after the kappacin:zinc treatment the number ofviable S. mutans in the biofilm had decreased to less than 0.5% ofpretreatment levels. Further, the viability of S. mutans in the biofilmdid not recover from the Kappacin:zinc treatment over the following 15days. To test the comparative efficacy of Kappacin and Kappacin:zinc a0.05% (w/v) solution of chlorhexidine digluconate was tested against S.mutans in the CDFF biofilm fermenter. A decrease in S. mutans cellviability of 48% was seen with a rapid recovery of cell viability suchthat 3.5 hour after treatment there was no significant difference topre-treatment viability.

Structural Analysis.

The amide region of a ¹H NMR spectrum of syntheticSer(P)¹⁴⁹κ-casein-A(138-158) recorded in 90% H₂O/10% D₂O solution showsthat the amide resonances are not well dispersed, occurring in a 0.6 ppmregion extending from about δ8.15 to δ8.75. This is characteristic ofpeptides in a ‘random-coil’ conformation. Addition of 5% (v/v) TFEresulted in a change of chemical shift for some of the resonances and ageneral broadening of the peaks. The broadening of the peaks is a resultof chemical exchange in which peptide molecules exist in twoenvironments, the aqueous solution or the more apolar environment of theTFE micelles. The peptide molecules exchange their environments at arate comparable to the difference in amide chemical shift in the twoenvironments. As more TFE was added to the sample the peptide becamepreferentially associated with the apolar TFE environment and the rateof exchange with the aqueous phase slowed. These changes are associatedwith further changes in amide chemical shifts and a general sharpeningof the NMR resonances. However, the range of chemical shifts is stillrelatively small with a range of 0.6 ppm from about δ8.1 to δ8.7indicating that the peptides are still in a ‘random-coil’ conformation.The addition of a molar excess of calcium ions resulted in the amideresonances spreading over a range of 1.25 ppm from δ7.75 to δ9.0, arange characteristic of a peptide in a specific conformation.

Clinical Trial.

A commercial preparation of Kappacin-enriched CMP was used in adouble-bind, cross over, small-scale clinical antiplaque trial involving10 subjects. HPLC analysis of the preparation indicated that in a 10.0mg/ml solution there was 4.4 mg/ml of nonglycosylatedκ-casein-A(106-169), 1.9 mg/ml of nonglycosylated κ-casein-B(106-169)and 3.0 mg/ml of glycosylated κ-casein(106-169). Based on a calculatedaverage molecular weight for glycosylated κ-casein(106-169) of 7500,there was a concentration of ˜1.33 mM of all forms of κ-casein(106-169)in the preparation, therefore there was a Zn:CMP ratio of ˜15:1.

After four days with the water (control) mouthrinse as the only form oforal hygiene the mean whole mouth Silness and Loe Plaque Index Score was178.9±33.5; when only considering the posterior teeth a mean PlaqueIndex Score of 85.9±14.4 was obtained. Compared with the water controlthe Kappacin mouthrinse resulted in a decrease in the mean Plaque IndexScore of posterior teeth of 7%, the ZnCl mouthrinse caused a 9% decreasewhilst the Zn:Kappacin treatment caused a decrease of 21% (FIG. 6). TheZnCl₂ mouthrinse significantly (P<0.05) reduced plaque accumulationrelative to the water treatment, as determined by a Wilcoxon rank testconsidering the posterior teeth plaque index scores. The Kappacinmouthrinse was not significantly different to the water control. TheZn:Kappacin mouthrinse significantly (P<0.05) reduced plaqueaccumulation relative to all other treatments.

The distribution of plaque scores on the posterior teeth also changedwith the type of mouthrinse used. The Kappacin:zinc containingmouthrinse resulted in only 47% of surfaces having a plaque index scoreof 2 or above compared with the water mouthrinse where 78% of toothsurfaces had a score of 2 or more (FIG. 7).

Discussion

The nonglycosylated, phosphorylated forms of the caseinomacropeptide[κ-casein(106-169)], (Kappacin) have been shown to have antibacterialactivity against both Gram-negative and Gram-positive oral bacteria atacidic pH. Of the six known genetic variants κ-casein-A(106-169) andκ-casein-B(106-169) are, by far, the most abundant forms. We havepreviously shown that κ-casein-A(106-169) had better antibacterialactivity than κ-casein-B(106-169), that the antibacterial activity ofκ-casein-A(106-169) could be localised to residues 138-158 and thatphosphorylation of Ser¹⁴⁹ was essential for activity (Malkoski et al.,2001, WO99/26971). To determine if the lower antibacterial activity ofκ-casein-B(106-169) was due to the hydrophilic to hydrophobic amino acidsubstitution within the 138-158 region (Asp¹⁴⁸ in variant A to Ala¹⁴⁸ invariant B) the activity of the synthetic peptideSer(P)¹⁴⁹κ-casein-B(138-158) was determined. The MIC for syntheticSer(P)¹⁴⁹κ-casein-B(138-158) against S. mutans at pH 6.28 was 44 μMwhich compared with the previous study showing the MIC of syntheticSer(P)¹⁴⁹κ-casein-A(138-158) under identical conditions was 26 μM(Malkoski et al., 2001). Therefore the difference in amino acid sequenceof the active region is likely to account for most, if not all, of thedifference in activity of the A and B genetic variants ofκ-casein(106-169) against S. mutans at pH 6.28.

At neutral pH (7.20) neither of the nonglycosylated, phosphorylatedκ-casein(106-169) genetic variants had antibacterial activity againstthe indicator species S. mutans (Table 2). The addition of the divalentmetal cation zinc in a 1:1 ratio with κ-casein-A(106-169) produced anantibacterial effect against S. mutans with an MIC of 161 μM which waslower than that seen for zinc alone (Table 2, FIG. 3). The combinationof zinc with κ-casein-B(106-169) in a 1:1 ratio did not produce an MICthat was lower than that for zinc alone, however at sub-MIC levels thiscombination had some growth inhibitory activity that was not detectedwith zinc or κ-casein-B(106-169) alone at the same concentrations (FIG.3). The combination of zinc and the synthetic peptideSer(P)¹⁴⁹κ-casein-A(138-158) in a 1:1 ratio had a similar MIC to thatseen with zinc:κ-casein-A(106-169), indicating that the divalent metalions may interact with this region of the peptide. To determine if thisenhanced activity at neutral pH was due to the antibacterial activity ofzinc or whether it was due to a conformational change in the peptidecalcium, a divalent metal cation with no antibacterial activity, wastested with CMP-derived peptides. The addition calcium in a 1:1 ratiowith κ-casein-A(106-169) produced an antibacterial effect against S.mutans with an MIC of 248 μM (Table 2). Calcium alone had no effect onS. mutans growth at concentrations up to 1 mM. No antibacterial effectwas detected by combining calcium with κ-casein-B(106-169) (Table 2).This suggests that the presence of the divalent metal cation is helpingto potentiate the activity of κ-casein-A(106-169) at neutral pH possiblyby modifying the structure of the peptide. The most efficacious ratio ofdivalent metal cation to κ-casein-A(106-169) was determined to be 2:1using calcium (FIG. 4) which was consistent with the results ofScatchard analysis indicating that κ-casein-A(106-169) specificallybinds two divalent metal cations (either calcium or zinc; FIG. 5).

In vivo oral bacteria are found as dental plaque, a biofilm attached tothe hard tissues (teeth). S. mutans was grown as a biofilm in a constantdepth film fermenter in a growth medium containing free sucrose to moreclosely simulate conditions found in the oral cavity. To accuratelydetermine the antimicrobial activity of an agent it should be tested ina biofilm model (Wilson, 1996). The constant depth film fermenterprovides a sophisticated means of reproducibly producing large numbersof biofilms that can then be used to quantitate the effects ofantimicrobial agents that gives predictive results for antiplaqueactivity (Hope and Wilson, 2003; Shu et al., 2003; Wilson, 1996;Wimpenny et al., 1989). The addition of Kappacin or Kappacin:zincsolutions to the S. mutans biofilm resulted in a dramatic decrease ofviable cells. Interestingly this decrease in bacterial cell numbers wassustained for a long period of time, indicating that Kappacin mayfunction more efficiently against biofilm bacteria than planktonicbacteria. In comparison a 0.05% solution of chlorhexidine, a recognisedantiplaque additive to mouthrinses, had a lesser effect on S. mutansviability and a less sustained effect.

Kappacin is an unusual antibacterial peptide in that it contains a highproportion of negatively charged amino acids. The Pi ofκ-casein-A(106-169) is 3.9 and over the pH range 5 to 8 there is littlechange in the charge of the molecule, which is approximately −7.

Structural analysis of the synthetic peptide κ-casein-A(138-158)indicated that it will interact with apolar phases, such as thebacterial cell membrane, and that in the presence of excess calcium ionsadopts a specific conformation in that environment. These conclusionsare consistent with the work of both Smith et al. (2002), who determinedthe structure of glycosylated and nonglycosylated CMP in the absence ofcalcium and found it to be largely random, flexible structure, andPlowman, (1997) who found that this region of CMP had a propensity toform an amphipathic α-helix, in the presence of TFE.

A commercial preparation of Kappacin, made from casein, was used in aclinical antiplaque trial. In this preparation the nonglycosylated formsof CMP (Kappacin) accounted for 63% of the dry weight, of which 70% wasgenetic variant A and 30% was variant B. The Kappacin-zinc combinationmouthrinse was significantly more effective in controlling supragingivaldental plaque when used as the only oral hygiene procedure thanmouthrinses containing Kappacin or zinc alone, suggesting a synergisticeffect. Zinc citrate has previously been shown in a double blindcrossover trial to significantly reduce the plaque accumulation insubjects by 5-8% using the Turesky plaque index (Addy et al., 1980). Thezinc chloride mouthrinse produced similar results in this study, wheremean whole mouth plaque index scores, and mean posterior teeth plaqueindex scores decreased by 6% and 9%, respectively. Giertsen et al.(1988) showed that the use of zinc chloride as a mouthrinse resulted ina significant increase of 9% in the tooth surfaces with no plaque, usingthe Silness and Loe plaque index, compared with a water control.Although the Kappacin mouthrinse showed a reduction in supragingivalplaque of posterior teeth of 7% when compared with the water, thisreduction in plaque was not statistically different to the control. Inthe current study the Silness and Loe plaque index was used as changesin plaque thickness, especially along the gingival margin, are morereadily observed than changes in plaque distribution over the surfacesof teeth. Therefore due to short duration of the trial (5 days) and thesmall number of subjects, the Silness and Loe index was deemed to be themost appropriate plaque scoring system. It has also been shown thatunstained plaque scores correlate much higher than stained scores withgingivitis, dry and wet plaque weight (Loesche and Green, 1972).

Salts of zinc and tin have long been recognised as having antibacterialactivity and a relatively high safety profile (Moran et al., 2000). Zincis believed to exert its antibacterial effect by inhibiting membranetransport and metabolic processes, including glycolysis, throughinteractions with enzymes that contain active thiol groups (Cummins andCreeth, 1992, Opperman and Rolla, 1980; Opperman et al., 1980). Theadsorption of zinc into plaque bacteria initially involves electrostaticinteractions with cell surface proteins followed by their subsequenttransport into the cell. The plaque inhibiting effect is thought to be along acting bacteriostatic effect on plaque microorganisms through theretention of the ions in dental plaque and the oral cavity after rinsing(Giertsen et al., 1988). Zinc salts have been used in toothpastes andmouthrinses in combination with the antimicrobial agents triclosan,chlorhexidine and sanguinarine. These have been shown to have asynergistic antibacterial action (Giertsen et al., 1988; Moran et al.,2000).

Treatment with the Kappacin-zinc containing mouthrinse resulted in adecrease of 21% in the posterior teeth plaque index scores (FIG. 6).These results are comparable to chlorhexidine mouthrinses. Chlorhexidineis considered to be the most effective anti-plaque and anti-gingivitiscompound so far tested. However side effects from the use ofchlorhexidine, including extrinsic staining of teeth and restorations,taste distortion, and brown staining of tongue, limit its long term use(Elderidge et al., 1998).

It is well accepted that the accumulation of supragingival plaque over aperiod of time is associated with the development of gingivitis, andinitiation of periodontitis and that supragingival plaque control aloneis sufficient to resolve gingivitis (Corbet and Davies, 1993). Theresults of this study indicate that the combination of the naturalpeptide Kappacin with zinc ions may produce a mouthrinse with efficacyin supragingival plaque control.

Proposed Formulations Including the Composition of the Present Invention

Formulation 1

Ingredient % w/w Dicalcium phosphate dihydrate 50.0 Glycerol 20.0 Sodiumcarboxymethyl cellulose 1.0 Sodium lauryl sulphate 1.5 Sodium lauroylsarconisate 0.5 Flavour 1.0 Sodium saccharin 0.1 Chlorhexidine gluconate0.01 Dextranase 0.01 Composition of present invention 1.0 Water balanceFormulation 2

Ingredient % w/w Dicalcium phosphate dihydrate 50.0 Sorbitol 10.0Glycerol 10.0 Sodium carboxymethyl cellulose 1.0 Sodium lauryl sulphate1.5 Sodium lauroyl sarconisate 0.5 Flavour 1.0 Sodium saccharin 0.1Sodium monofluorophosphate 0.3 Chlorhexidine gluconate 0.01 Dextranase0.01 Composition of present invention 2.0 Water balanceFormulation 3

Ingredient % w/w Dicalcium phosphate dihydrate 50.0 Sorbitol 10.0Glycerol 10.0 Sodium carboxymethyl cellulose 1.0 Lauroyl diethanolamide1.0 Sucrose monolaurate 2.0 Flavour 1.0 Sodium saccharin 0.1 Sodiummonofluorophosphate 0.3 Chlorhexidine gluconate 0.01 Dextranase 0.01Composition of present invention 5.0 Water balanceFormulation 4

Ingredient % w/w Sorbitol 10.0 Irish moss 1.0 Sodium Hydroxide (50%) 1.0Gantrez 19.0 Water (deionised) 2.69 Sodium monofluorophosphate 0.76Sodium saccharin 0.3 Pyrophosphate 2.0 Hydrated alumina 48.0 Flavour oil0.95 Composition of present invention 1.0 Water balanceFormulation 5

Ingredient % w/w Sodium polyacrylate 50.0 Sorbitol 10.0 Glycerol 20.0Sodium saccharin 0.1 Sodium monofluorophosphate 0.3 Chlorhexidinegluconate 0.01 Ethanol 3.0 Composition of present invention 2.0 Linolicacid 0.05 Water balanceProposed Mouthwash FormulationsFormulation 1

Ingredient % w/w Ethanol 20.0 Flavour 1.0 Sodium saccharin 0.1 Sodiummonofluorophosphate 0.3 Chlorhexidine gluconate 0.01 Lauroyldiethanolamide 0.3 Composition of present invention 2.0 Water balanceFormulation 2

Ingredient % w/w Gantrez S-97 2.5 Glycerine 10.0 Flavour oil 0.4 Sodiummonofluorophosphate 0.05 Chlorhexidine gluconate 0.01 Lauroyldiethanolamide 0.2 Composition of present invention 2.0 Water balanceProposed Lozenge Formulation

Ingredient % w/w Sugar 75-80 Corn syrup  1-20 Flavour oil 1-2 NaF0.01-0.05 Composition of present invention 3.0 Mg stearate 1-5 WaterbalanceProposed Gingival Massage Cream Formulation

Ingredient % w/w White petrolatum 8.0 Propylene glycol 4.0 Stearylalcohol 8.0 Polyethylene Glycol 4000 25.0 Polyethylene Glycol 400 37.0Sucrose monostearate 0.5 Chlorohexidine gluconate 0.1 Composition ofpresent invention 3.0 Water balanceProposed Chewing Gum Formulation

Ingredient % w/w Gum base 30.0 Calcium carbonate 2.0 Crystallinesorbitol 53.0 Glycerine 0.5 Flavour oil 0.1 Composition of presentinvention 2.0 Water balance

All publications mentioned in this specification are herein incorporatedby reference. Any discussion of documents, acts, materials, devices,articles or the like which has been included in the presentspecification is solely for the purpose of providing a context for thepresent invention. It is not to be taken as an admission that any or allof these matters form part of the prior art base or were common generalknowledge in the field relevant to the present invention as it existedin Australia or elsewhere before the priority date of each claim of thisapplication.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

REFERENCES

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1. An antimicrobial composition, the composition comprising an aqueoussolvent and having a divalent cation and a peptide dissolved therein,wherein the divalent cation is added to the solvent such thatsubstantially all of the divalent cation is dissolved in the solvent,wherein the divalent cation is a Ca²⁺ or a Zn²⁺ ion, and wherein thepeptide is non-glycosylated, has a length of less than about 100 aminoacids, and comprises an amino acid sequence selected from the groupconsisting of: Ala Val Glu Ser Thr Val Ala Thr Leu Glu Ala X Pro Glu ValIle Glu Ser Pro Pro Glu (SEQ ID NO:1); and Ala Val Glu Ser Thr Val AlaThr Leu Glu Asp X Pro Glu Val Ile Glu Ser Pro Pro Glu (SEQ ID NO:2),wherein amino acid residue X is a phosphoseryl residue.
 2. Theantimicrobial composition according to claim 1 wherein the peptide has alength of less than about 70 amino acids.
 3. The antimicrobialcomposition according to claim 1 wherein the peptide comprises the aminoacid sequence Ala Val Glu Ser Thr Val Ala Thr Leu Glu Ala X Pro Glu ValIle Glu Ser Pro Pro Glu (SEQ ID NO:1), wherein amino acid residue X is aphosphoseryl residue.
 4. The antimicrobial composition according toclaim 1 wherein the peptide comprises an amino acid sequence selectedfrom the group consisting of: Met Ala Ile Pro Pro Lys Lys Asn Gln AspLys Thr Glu Ile Pro Thr Ile Asn Thr Ile Ala Ser Gly Glu Pro Thr Ser ThrPro Thr Ile Glu Ala Val Glu Ser Thr Val Ala Thr Leu Glu Ala X Pro GluVal Ile Glu Ser Pro Pro Glu Ile Asn Thr Val Gln Val Thr Ser Thr Ala Val(SEQ ID NO:3); Met Ala Ile Pro Pro Lys Lys Asn Gln Asp Lys Thr Glu IlePro Thr Ile Asn Thr Ile Ala X Gly Glu Pro Thr Ser Thr Pro Thr Ile GluAla Val Glu Ser Thr Val Ala Thr Leu Glu Ala X Pro Glu Val Ile Glu SerPro Pro Glu Ile Asn Thr Val Gln Val Thr Ser Thr Ala Val (SEQ ID NO:4);Met Ala Ile Pro Pro Lys Lys Asn Gln Asp Lys Thr Glu Ile Pro Thr Ile AsnThr Ile Ala Ser Gly Glu Pro Thr Ser Thr Pro Thr Thr Glu Ala Val Glu SerThr Val Ala Thr Leu Glu Asp X Pro Glu Val Ile Glu Ser Pro Pro Glu IleAsn Thr Val Gln Val Thr Ser Thr Ala Val (SEQ ID NO. 5); Met Ala Ile ProPro Lys Lys Asn Gln Asp Lys Thr Glu Ile Pro Thr Ile Asn Thr Ile Ala XGly Glu Pro Thr Ser Thr Pro Thr Thr Glu Ala Val Glu Ser Thr Val Ala ThrLeu Glu Asp X Pro Glu Val Ile Glu Ser Pro Pro Glu Ile Asn Thr Val GlnVal Thr Ser Thr Ala Val (SEQ ID NO. 6); Thr Glu Ile Pro Thr Ile Asn ThrIle Ala Ser Gly Glu Pro Thr Ser Thr Pro Thr Ile Glu Ala Val Glu Ser ThrVal Ala Thr Leu Glu Ala X Pro Glu Val Ile Glu Ser Pro Pro Glu Ile AsnThr Val Gln Val Thr Ser Thr Ala Val (SEQ ID NO. 7); Thr Glu Ile Pro ThrIle Asn Thr Ile Ala X Gly Glu Pro Thr Ser Thr Pro Thr Ile Glu Ala ValGlu Ser Thr Val Ala Thr Leu Glu Ala X Pro Glu Val Ile Glu Ser Pro ProGlu Ile Asn Thr Val Gln Val Thr Ser Thr Ala Val (SEQ ID NO. 8); Thr GluIle Pro Thr Ile Asn Thr Ile Ala Ser Gly Glu Pro Thr Ser Thr Pro Thr ThrGlu Ala Val Glu Ser Thr Val Ala Thr Leu Glu Asp X Pro Glu Val Ile GluSer Pro Pro Glu Ile Asn Thr Val Gln Val Thr Ser Thr Ala Val (SEQ ID NO.9); and Thr Glu Ile Pro Thr Ile Asn Thr Ile Ala X Gly Glu Pro Thr SerThr Pro Thr Thr Glu Ala Val Glu Ser Thr Val Ala Thr Leu Glu Asp X ProGlu Val Ile Glu Ser Pro Pro Glu Ile Asn Thr Val Gln Val Thr Ser Thr AlaVal (SEQ ID NO. 10), wherein amino acid residue X is a phosphoserylresidue.
 5. The antimicrobial composition according to claim 4, whereinthe divalent cation is a Ca²⁺ ion.
 6. The antimicrobial compositionaccording to claim 4, wherein the divalent cation is Zn²⁺.
 7. Theantimicrobial composition according to claim 1 wherein the compositionhas a molar ratio of the divalent cation to the peptide in the range of0.5-15.0:1.0.
 8. The antimicrobial composition according to claim 7wherein the molar ratio of the divalent cation to the peptide is in therange of 0.5:1.0 to 4.0:1.0.
 9. The antimicrobial composition accordingto claim 8 wherein the molar ratio of the divalent cation to the peptideis in the range of 1.0:1.0 to 4.0:1.0.
 10. The antimicrobial compositionaccording to claim 9 wherein the molar ratio of the divalent cation tothe peptide is in the range of 1.0:1.0 to 2.0:1.0.
 11. A pharmaceuticalcomposition comprising a composition according to claim 1 and apharmaceutically acceptable carrier.
 12. A method of treatment,comprising: a) administering to a subject a therapeutically effectiveamount of a formulation comprising: an aqueous solvent and having adivalent cation and a peptide dissolved therein, wherein the divalentcation is added to the solvent such that substantially all of thedivalent cation is dissolved in the solvent, wherein the divalent cationis a Ca²⁺ or a Zn²⁺ ion, and wherein the peptide is non-glycosylated,has a length of less than about 100 amino acids, and comprises an aminoacid sequence selected from the group consisting of: Ala Val Glu Ser ThrVal Ala Thr Leu Glu Ala X Pro Glu Val Ile Glu Ser Pro Pro Glu (SEQ IDNO:1); and Ala Val Glu Ser Thr Val Ala Thr Leu Glu Asp X Pro Glu Val IleGlu Ser Pro Pro Glu (SEQ ID NO:2), wherein amino acid residue X is aphosphoseryl residue ;and b) allowing the formulation to act on thesubject in a manner which prevents a disease selected from the groupconsisting of dental caries and periodontal disease.
 13. The method ofclaim 12, wherein the administering is directly to the teeth or gums ofthe subject.
 14. A method of claim 12, wherein the administering is bytopical administration.
 15. An antimicrobial composition according toclaim 1 wherein the divalent cation is Zn²⁺.
 16. An antimicrobialcomposition according to claim 1 wherein the divalent cation is Ca²⁺.17. The antimicrobial composition according to claim 1 wherein thepeptide comprises the amino acid sequence Ala Val Glu Ser Thr Val AlaThr Leu Glu Asp X Pro Glu Val Ile Glu Ser Pro Pro Glu (SEQ ID NO:2),wherein amino acid residue X is a phosphoseryl residue.